sDevelopment and characterization of a walnut protein hydrolysate-Fe-vitamin C complex: Enhancing iron bioavailability through phytase-assisted modification

In this study, walnut protein was treated with phytase and Alcalase to produce walnut protein hydrolysate (WPHP). Phytase hydrolysis reduced phytic acid‑iron interactions, exposing peptide binding sites and enabling strong Fe²⁺ chelation rate of 80.78 ± 0.58 %. Vitamin C was subsequently added to form WPHP-Fe-vitamin C complex, which was stabilized through Fe-carboxyl and Fe‑carbonyl coordination and hydrogen bonding, accompanied by structural reorganization. The complex exhibited a porous microstructure, particle size of 345.76 nm, solubility of 90.22 % at pH 7.0, and stabilized under varying pH, ionic, and thermal conditions. Compared with WPHP-Fe, the complex showed higher iron retention. Caco-2 assays revealed that vitamin C mitigated the inhibitory effect of phytic acid and significantly enhanced Fe²⁺ uptake via the DMT1 pathway, reaching 9.47 ± 0.13 μg/well at 120 min. These results highlight the complementary roles of phytase and vitamin C and support the complex as a peptide-based iron supplement.